5-Carboxy-phenylenebis(carbonylimino)benzene carboxylic and dicarboxylic acids and salts

ABSTRACT

5-Carboxy-phenylenebis(carbonylimino)benzene carboxylic and dicarboxylic acids and salts thereof useful as complement inhibitors.

DESCRIPTION OF THE INVENTION

This invention is concerned with compounds of the formula: ##STR1##wherein R is selected from the group consisting of hydrogen and alkalimetal; and with the proviso that the terminal phenyls may only bedi-substituted.

These novel compounds are useful as inhibitors of the complement systemof warm-blooded animals.

A preferred embodiment of the instant invention consists of thosecompounds wherein R is hydrogen. Another preferred embodiment consistsof those compounds wherein R is selected from the group consisting ofalkali metal.

A most preferred embodiment of the two preferred embodiments consists ofthose compounds wherein the terminal phenyls are disubstituted at thecarbon 3,4-position.

The compounds of the present invention may be prepared by treating anappropriate mono or disubstituted carboxylic acid amino benzene(neutralized in aqueous medium) with 1,3,5-benzenetricarboxylic acidchloride in the presence of sodium carbonate for up to 18 hours. Afteracidification with hydrochloric acid, the product is isolated byconventional extraction procedures from ethanol and water.

The term "complement" refers to a complex group of proteins in bodyfluids that, working together with antibodies or other factors, play animportant role as mediators of immune, allergic, immunochemical and/orimmunopathological reactions. The reactions in which complementparticipates take place in blood serum or in other body fluids, andhence are considered to be humoral reactions.

With regard to human blood, there are at present more than 11 proteinsin the complement system. These complement proteins are designated bythe letter C and by number: C1, C2, C3 and so on up to C9. Thecomplement protein C1 is actually an assembly of subunits designatedC1q, C1r and C1s. The numbers assigned to the complement proteinsreflect the sequence in which they become active, with the exception ofcomplement protein C4, which reacts after C1 and before C2. Thenumerical assignments for the proteins in the complement system weremade before the reaction sequence was fully understood. A more detaileddiscussion of the complement system and its role in body processes canbe found in, for example, Bull. World Health Org., 39, 935-938 (1968);Ann. Rev. Medicine, 19, 1-24 (1968); The John Hopkins Med. J., 128,57-74 (1971); Harvey Lectures, 66, 75-104 (1972); The New EnglandJournal of Medicine, 287, 452-454; 489-495; 545-549; 592-596; 642-646(1972); Scientific American, 229, (No. 5), 54-66 (1973); FederationProceedings, 32, 134-137 (1973); Medical World News, October 11, 1974,pp. 53-58; 64-66; J. Allergy Clin. Immunol., 53, 298-302 (1974); ColdSpring Harbor Conf. Cell Proliferation 2/Proteases Biol. Control/229-241(1975); Annals of Internal Medicine, 84, 580-593 (1976); "Complement:Mechanisms and Functions, " Prentice-Hall, Englewood Cliffs, N. J.(1976).

The complement system can be considered to consist of three sub-systems:(1) a recognition unit (C1q) which enables it to combine with antibodymolecules that have detected a foreign invader; (2) an activation unit(C1r, C1s, C2, C4, C3) which prepares a site on the neighboringmembrane; and (3) and attack unit (C5, C6, C7, C8 and C9) which createsa "hole" in the membrane. The membrane attack unit is non-specific; itdestroys invaders only because it is generated in their neighborhood. Inorder to minimize damage to the host's own cells, its activity must belimited in time. This limitation is accomplished partly by thespontaneous decay of activated complement and partly by interference byinhibitors and destructive enzymes. The control of complement, however,is not perfect, and there are times when damage is done to the host'scells. Immunity is therefore a double-edged sword.

Activation of the complement system also accelerates blood clotting.This action comes about by way of the complement-mediated release of aclotting factor from platelets. The biologically active complementfragments and complexes can become involved in reations that damage thehost's cells, and these pathogenic reactions can result in thedevelopment of immune-complex diseases. For example, in some forms ofnephritis, complement damages the basal membrane of the kidney,resulting in the escape of protein from the blood into the urine. Thedisease disseminated lupus erythematosus belongs in this category; itssymptoms include nephritis, visceral lesions and skin eruptions. Thetreatment of diphtheria or tetanus with the injection of large amountsof antitoxin sometimes results in serum sickness, an immune-complexdisease. Rheumatoid arthritis also involves immune complexes. Likedisseminated lupus erythematosus, it is an autoimmune disease in whichthe disease symptoms are caused by pathological effects of the immunesystem in the host's tissues. In summary, the complement system has beenshown to be involved with inflammation, coagulation, fibrinolysis,antibody-antigen reactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteinsare involved in a series of reactions which may lead to irreversiblemembrane damage if they occur in the vicinity of biological membranes.Thus, while complement constitutes a part of the body's defensemechanism against infection it also results in inflammation and tissuedamage in the immunopathological process. The nature of certain of thecomplement proteins, suggestions regarding the mode of complementbinding to biological membranes and the manner in which complementeffects membrane damage are discussed in Annual Review in Biochmeistry,388, 389 (1969).

A variety of substances have been disclosed as inhibiting the complementsystem, i.e., as complement inhibitors. For example, the compounds3,3'-ureylenebis-[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)]benzenesulfonicacid, tetrasodium salt (chlorazol fast pink), heparin and a sulphateddextran have been reported to have an anticomplementary effect, BritishJournal of Experimental Pathology, 33, 327-339 (1952). The compound8-(3-benzamido-4-methylbenzamido)naphthalene-1, -3,5-trisulfonic acid(Suramin) is described as a competitive inhibitor of the complementsystem, Clin. Exp. Immunol., 10, 127-138 (1972). German Pat. No.2,254,893 or South African Pat. No. 727,923 discloses certain1-(diphenylmethyl)-4-(3-phenylallyl)piperazines useful as complementinhibitors. Other chemical compounds having complement inhibitingactivity are disclosed in, for example, Journal of Medicinal Chemistry,12, 415-419; 902-905; 1049-1052; 1053-1056 (1969); Canadian Journal ofBiochemistry, 47, 547-552 (1969); The Journal of Immunology, 93, 629-640(1964); The Journal of Immunology, 104, 279-288 (1970); The Journal ofImmunology, 106, 241-245 (1971); and The Journal of Immunology, 111,1061-1066 (1973).

It has been reported that the known complement inhibitorsepsilon-aminocaproic acid, Suramin and tranexamic acid all have beenused with success in the treatment of hereditary angioneurotic edema, adisease state resulting from an inherited deficiency or lack of functionof the serum inhibitor of the activated first component of complement(C1 inhibitor), The New England Journal of Medicine, 286, 808-812(1972).

EXAMPLE 1 4,4'-[5-Carboxy-1,3-phenylenebis(carbonylimino)]-diphthalicacid pentasodium salt

A 2.5 g portion of 4-amino phthalic acid stirred in 100 ml of water isneutralized to pH 7.2 with sodium hydroxide then 1.47 g of sodiumcarbonate is added. To this solution is added dropwise 1.21 g of1,3,5-benzenetricarboxylic acid chloride with vigorous stirring.Stirring is continued for 18 hours, then the reaction mixture isconcentrated, dissolved in 60 ml of water and acidified withhydrochloric acid. The precipitate is filtered and washed with ethanoland ether to yield 2.8 g of product. This product is dissolved in 50 mlof water, is neutralized and fractionally precipitated with ethanol 6times. The final crop, 523 mg, is identified as the product of theExample.

EXAMPLE 2

    ______________________________________                                        Preparation of Compressed Tablet                                              Ingredient           mg/Tablet                                                ______________________________________                                        Active Compound      0.5 - 500                                                Dibasic Calcium Phosphate N.F.                                                                     qs                                                       Starch USP           40                                                       Modified Starch      10                                                       Magnesium Stearate USP                                                                             1 - 5                                                    ______________________________________                                    

EXAMPLE 3

    ______________________________________                                        Preparation of Compressed Tablet - Sustained Action                           Ingredient           mg/Tablet                                                ______________________________________                                        Active Compound      0.5 - 500 (as acid                                       as Aluminum Lake*, Micronized                                                                      equivalent)                                              Dibasic Calcium Phosphate N.F.                                                                     qs                                                       Alginic Acid         20                                                       Starch USP           35                                                       Magnesium Stearate USP                                                                             1 - 10                                                   ______________________________________                                         *Complement inhibitor plus aluminum sulfate yields aluminum complement        inhibitor. Complement inhibitor content in aluminum lake ranges from 5 -      30%.                                                                     

EXAMPLE 4

    ______________________________________                                        Preparation of Hard Shell Capsule                                             Ingredient           mg/Capsule                                               ______________________________________                                        Active Compound      0.5 - 500                                                Lactose, Spray Dried qs                                                       Magnesium Stearate   1 - 10                                                   ______________________________________                                    

EXAMPLE 5

    ______________________________________                                        Preparation of Oral Liquid (Syrup)                                            Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                 Liquid Sugar         75.0                                                     Methyl Paraben USP   0.18                                                     Propyl Paraben USP   0.02                                                     Flavoring Agent      qs                                                       Purified Water qs ad 100.0                                                    ______________________________________                                    

EXAMPLE 6

    ______________________________________                                        Preparation of Oral Liquid (Elixir)                                           Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                 Alcohol USP          12.5                                                     Glycerin USP         45.0                                                     Syrup USP            20.0                                                     Flavoring Agent      qs                                                       Purified Water qs ad 100.0                                                    ______________________________________                                    

EXAMPLE 7

    ______________________________________                                        Preparation of Oral Suspension (Syrup)                                        Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                 as Aluminum Lake, Micronized                                                                       (acid equivalent)                                        Polysorbate 80 USP   0.1                                                      Magnesium Aluminum Silicate,                                                  Colloidal            0.3                                                      Flavoring Agent      qs                                                       Methyl Paraben USP   0.18                                                     Propyl Paraben USP   0.02                                                     Liquid Sugar         75.0                                                     Purified Water qs ad 100.0                                                    ______________________________________                                    

EXAMPLE 8

    ______________________________________                                        Preparation of Injectable Solution                                            Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                 Benzyl Alcohol N.F.  0.9                                                      Water for Injection  100.0                                                    ______________________________________                                    

EXAMPLE 9

    ______________________________________                                        Preparation of Injectable Oil                                                 Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                 Benzyl Alcohol       1.5                                                      Sesame Oil qs ad     100.0                                                    ______________________________________                                    

EXAMPLE 10

    ______________________________________                                        Preparation of Intra-Articular Product                                        Ingredient           Amount                                                   ______________________________________                                        Active Compound      2 - 20 mg                                                NaCl (physiological saline)                                                                        0.9%                                                     Benzyl Alcohol       0.9%                                                     Sodium Carboxymethylcellulose                                                                      1 - 5%                                                   pH adjusted to 5.0 - 7.5                                                      Water for Injection qs ad                                                                          100%                                                     ______________________________________                                    

EXAMPLE 11

    ______________________________________                                        Preparation of Injectable Depo Suspension                                     Ingredient           % W/V                                                    ______________________________________                                        Active Compound      0.05 - 5                                                                      (acid equivalent)                                        Polysorbate 80 USP   0.2                                                      Polyethylene Glycol 4000 USP                                                                       3.0                                                      Sodium Chloride USP  0.8                                                      Benzyl Alcohol N.F.  0.9                                                      HCl to pH 6 - 8      qs                                                       Water for Injection qs ad                                                                          100.0                                                    ______________________________________                                    

The compounds of the present invention may be administered internally,e.g., orally, intra-articularly or parenterally, e.g., intra-articular,to a warm-blooded animal to inhibit complement in the body fluid of theanimal, such inhibition being useful in the amelioration or preventionof those reactions dependent upon the function of complement, such asinflammatory process and cell membrane damage induced byantigen-antibody complexes. A range of doses may be employed dependingon the mode of administration, the condition being treated and theparticular compound being used. For example, for intraveneous orsubcutaneous use from about 5 to about 50 mg/kg/day, or every 6 hoursfor more rapidly excreted salts, may be used. For intra-articular usefor large joints such as the knee, from about 2 to about 20 mg/joint perweek may be used, with proportionally smaller doses for smaller joints.The dosage range is to be adjusted to provide optimum therapeuticresponse in the warm-blooded animal being treated. In general, theamount of compound administered can vary over a wide range to providefrom about 5 mg/kg to about 100 mg/kg of body weight of animal per day.The usual daily dosage for a 70 kg subject may vary from about 350 mg toabout 3.5 g. Unit doses of the acid or salt can contain from about 0.5to about 500 mg.

While in general the sodium salts of the acids of the invention aresuitable for parenteral use, other salts may also be prepared, such asthose of primary amines, e.g., ethylamine; secondary amines, e.g.,diethylamine or diethanol amine; tertiary amines, e.g., pyridine ortriethylamine or 2-dimethylaminomethyl-dibenzofuran; aliphatic diamines,e.g., decamethylenediamine; and aromatic diamines, can be prepared. Someof these are soluble in water, others are soluble in saline solution,and still others are insoluble and can be used for purposes of preparingsuspensions for injection. Furthermore as well as the sodium salt, thoseof the alkali metals, such as potassium and lithium; of ammonia; and ofthe alkaline earth metals, such as calcium or magnesium, may beemployed. It will be apparent, therefore, that these salts embrace, ingeneral derivatives of salt-forming cations.

In therapeutic use, the compounds of this invention may be administeredin the form of conventional pharmaceutical compositions. Suchcompositions may be formulated so as to be suitable for oral orparenteral administration. The active ingredient may be combined inadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, i.e., oral or parenteral. The compounds canbe used in compositions such as tablets. Here, the principal activeingredient is mixed with conventional tabletting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate, gums, or similar materials as non-toxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action of the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate non-toxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration include suitableflavored emulsions with edible oils, such as, cottonseed oil, sesameoil, coconut oil, peanut oil, and the like, as well as elixirs andsimilar pharmaceutical vehicles. Sterile suspensions or solutions can beprepared for parenteral use. Isotonic preparations containing suitablepreservatives are also desirable for injection use.

The term dosage form, as described herein, refers to physically discreteunits suitable as unitary dosage for warm-blooded animal subjects, eachunit containging a predetermined quantity of active component calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical diluent, carrier or vehicle. The specificationfor the novel dosage forms of this invention are indicated bycharacteristics of the active component and the particular therapeuticeffect to be achieved or the limitations inherent in the art ofcompounding such an active component for therapeutic use in warm-bloodedanimals as disclosed in this specification. Examples of suitable oraldosage forms in accord with this invention are tablets, capsules, pills,powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls,ampules, vials, segregated multiples of any of the foregoing and otherforms as herein described.

The complement inhibiting activity of the compounds of this inventionhas been demonstrated by one or more of the following identified tests:(i) Test, Code 026 (C1 inhibitor) -- This test measures the ability ofactivated human C1 to destroy fluid phase human C2 in the presence of C4and appropriate dilutions of the test compound. An active inhibitorprotects C2 from C1 and C4; (ii) Test, Code 035 (C3-C9 inhibitor) --This test determines the ability of the late components of humancomplement (C3-C9) to lyse EAC 142 in the presence of appropriatedilutions of the test compound. An active inhibitor protects EAC 142from lysis by human C3-C9; (iii) Test, Code 036 (C-Shunt inhibitor) --In this test human erythrocytes rendered fragile are lysed in autologousserum via the shunt pathway activated by cobra venom factor in thepresence of appropriate dilutions of the test compound. Inhibition ofthe shunt pathway results in failure of lysis; (iv) Forssman VasculitisTest -- Here, the well known complement dependent lesion, Forssmanvasculitis, is produced in guinea pigs by intradermal injection ofrabbit anti-Forssman antiserum. The lesion is measured in terms ofdiameter, edema and hemorrhage and the extent to which a combined indexof these is inhibited by prior intraperitoneal injection of the testcompound at 200 mg/k is then reported, unless otherwise stated; (v)Forssman Shock Test -- Lethal shock is produced in guinea pigs by ani.v. injection of anti-Forssman antiserum and the harmonic mean deathtime of treated guinea pigs is compared with that of simultaneouscontrols; (vi) Complement Level Reduction Test -- In this test, theabove dosed guinea pigs, or others, are bled for serum and thecomplement level is determined in undiluted serum by the capillary tubemethod of U.S. Pat. No. 3,876,376 and compared to undosed control guineapigs; and (vii) Cap 50 Test -- Here, appropriate amounts of the testcompound are added to a pool of guinea pig serum in vitro, after whichthe undiluted serum capillary tube assay referred to above is run. Theconcentration of compound inhibiting 50% is reported.

With reference to Table I, guinea pigs weighing about 300 g were dosedintravenously (i.v.) or intraperitoneally (i.p.) with 200 mg/kg of thetest compound dissolved in saline and adjusted to pH 7-8. One hour afterdosing, the guinea pigs were decapitated, blood was collected and theserum separated. The serum was tested for whole complement using thecapillary tube assay. Percent inhibition was calculated by comparisonwith simultaneous controls. The results appear in Table I together withresults of tests, code 026, 035, 036, Cap. 50, % inhibition and Forssmanshock. Table I shows that the compounds of the invention possess highlysignificant in situ and in vivo, complement inhibiting activity inwarm-blooded animals.

Some of the compounds of the present invention have been found topossess anti-coagulant activity as well as complement inhibitingactivity. The in vitro anti-coagulant activity (AC) of the compounds ofthis invention has been demonstrated by the following test: Citratedsheep plasma (CSP) is added to various dilutions of test compound in aMicrotiter® plate, the CSP sample mixtures are then recalcified with anisotonic sheep red blood cell (RBC) suspension. The sheep RBC's, kept insuspension throughout the clotting incubation time, become enmeshed inthe fibrin matrix if a clots forms. Upon centrifugation of the plate,untrapped RBC's form buttons, the sizes of which correspond to thedegree of clot inhibition; this providing a measure of anti-coagulantactivity (AC). Sodium heparin is used as a positive control and activityis reported in wells appearing in Table I.

                  TABLE I                                                         ______________________________________                                        Biological Activities                                                                                        Shunt                                                         Cl      C-Late  Inhibition                                                    026*    035*    036*    AC*                                    Compound       Wells   Wells   Wells   Wells                                  ______________________________________                                        4,4'-[5-Carboxy-1,3-phen-                                                     ylenebis(carbonylimino)]-                                                                    +2**    N       N       ≦-2**                           diphthalic acid penta-                                                        sodium salt                                                                   ______________________________________                                         *Code designation for tests employed as referred herein.                      **Activity in wells a serial dilution assay. Higher well number indicates     higher activity. The serial dilutions are two-fold.                      

The computation of an Intrinsic Therapeutic Index (ITI) was devised tocorrelate the results, expressed in wells, obtained in the in vitro Code026 (C1 inhibitor) test and the in vitro anti-coagulant (AC) test into ameaningful value which would aid in the net evaluation of the activityof the compounds of this invention.

The ITI of a given compound may be defined as the antilogarithm of thelogarithmic (base 2) difference between the highest serial dilution inwells which is active in the Code 026 test and the highest serialdilution in wells providing activity in the anti-coagulant test. The ITIis thus a measure of the separation of anti-complement andanti-coagulant activities; the higher the numerical value the moretherapeutically useful the separation of activities.

The Intrinsic Therapeutic Index of the compounds of this invention arelisted in Table II.

                                      TABLE II                                    __________________________________________________________________________    Intrinsic Therapeutic Index                                                                   In Vitro Activity                                                             Complement        Logarithmic                                                 Inhibiting                                                                             Anti-Coagulant                                                                         Difference                                                                           Intrinsic                                            Activity (Wells)                                                                       Activity (Wells)                                                                       Expressed as                                                                         Therapeutic                          Compound        Code 026 AC       Wells  Index                                __________________________________________________________________________    4,4'-[5-Carboxy-1,3-phenylenebis-                                             (carbonylimino)]diphthalic acid                                                               +2       -2       +4     16                                   pentasodium salt                                                              __________________________________________________________________________

We claim:
 1. A compound of the formula: ##STR2## wherein R is selectedfrom the group consisting of hydrogen and alkali metal; and with theproviso that the terminal phenyls may only be di-substituted.
 2. Acompound according to claim 1, wherein R is hydrogen.
 3. A compoundaccording to claim 1, wherein R is selected from the group consisting ofalkali metal.
 4. A compound according to claim 3, wherein the terminalphenyls are di-substituted at the carbon 3,4-position.
 5. The compoundaccording to claim 4,4,4'[5carboxy-1,3-phenylenebis(carbonylimino)]diphthalic acidpentasodium salt.
 6. A method of inhibiting the complement system in abody fluid which comprises subjecting said body fluid to the action ofan effective complement inhibiting amount of a compound of the formula:##STR3## wherein R is selected from the group consisting of hydrogen andalkali metal; and with the proviso that the terminal phenyls may only bedi-substituted.
 7. A method according to claim 6, wherein said compoundis 4,4'-[5-carboxy-1,3-phenylenebis(carbonylimino)]diphthalic acidpentasodium salt.
 8. A method according to claim 6, wherein said bodyfluid is blood serum.
 9. A method of inhibiting the complement system ina warm-blooded animal which comprises internally administering to saidanimal an effective complement inhibiting amount of a compound of theformula: ##STR4## wherein R is selected from the group consisting ofhydrogen and alkali metal; and with the proviso that the terminalphenyls may only be di-substituted.
 10. A method according to claim 9,wherein said compound is4,4'-[5-carboxy-1,3-phenylenebis(carbonylimino)]diphthalic acidpentasodium salt.
 11. A method according to claim 9, wherein saidadministering is intra-articularly.